A basic feature of a bioelectronic device is the immobilization of a biomaterial onto a conductive or semi-conductive support, and the electronic transduction of the biological functions associated with the biological substrates.
A biosensor is an analytical device incorporating biological and chemical sensing elements, either intimately connected to or integrated with a suitable transducer, which enables the conversion of concentrations of specific chemicals into electronic signals. A majority of biosensors produced thus far have incorporated enzymes as biological sensing elements (1). The electronic transduction of the enzyme-substrate interactions may also provide an analytical means to detect a respective substrate. The chemical means to assembly the enzymes on conductive or semi-conductive supports include the immobilization thereof on a substrate by means of self-assembling monolayers or thin films, polymer layers, membranes, carbon paste or sol-gel materials.
A specific class of enzymes which have been proposed for the use in analytical biochemical methods are redox enzymes. A redox reaction involves the transfer of electrons from the enzyme to the analyte—in a reduction reaction, or from the analyte to the enzyme in an oxidation reaction. If there is an electrical communication between the redox center of the enzyme molecules and the electrode material, there is an electrical charge flow which can serve as an indication of the presence of the analyte and the extent of charge flow may serve to measure the analyte's concentration. Alternatively, the determination may be based on the measurement of a product of the reaction by non-electrochemical means, e.g. by HPLC.
The direct electron transfer between the enzyme redox center and the electrode is limited, since the redox center is sterically insulated by the protein matrices. Consequently, the electrical communication between the redox enzymes and the electrodes may be established by an electron mediator group, often also termed “electron relay” (2), or by immobilizing the redox-proteins in electroactive polymers (3).
One of the attractive applications of bioelectrocatalytic electrodes is the development of biofuel cell assemblies. The biofuel cell utilizes biocatalysts for the conversion of chemical energy into electrical energy. Many organic substrates undergo combustion in oxygen or are oxidized with the release of energy. Methanol and glucose are abundant raw materials that can be used as biofuels which undergo oxidation, and molecular oxygen or hydrogen peroxide can act as the oxidizer.
For example, in a classical fuel cell where methanol is used as the fuel, the electro-oxidation of methanol at the anode can be represented by:CH3OH+H2O→CO2+6H++6e−,
and the electro-reduction of oxygen at the cathode can be represented by:O2+4H++4e−→2H2O.
Protons generated at the anode are transported to the cathode. A flow of current is sustained by a flow of ions through the membrane separating the cell into cathodic and anodic compartments and a flow of electrons through the external load.
An example of a biofuel cell assembly based on the bioelectrocatalytic oxidation of glucose by O2 (4) is showed schematically in FIG. 1. The cell consists of two electrodes, where the anode is functionalized by a surface-reconstituted glucose oxidase (GOx) monolayer and the cathode is modified with an integrated biocatalytic construction composed of cytochrome c (Cyt c) and cytochrome oxidase (COx). At the GOx monolayer-functionalyzed electrode, bioelectrocatalyzed oxidation of glucose to gluconic acid occurs, whereas at the Cyt c/COx layered electrode the reduction of O2 to water takes place. The GOx layer is generated by the reconstitution of apo-GOx (GOx without its FAD cofactor) on amino-FAD covalently linked to a pyrroloquinolino quinone (PQQ) monolayer. The PQQ unit acts as an electron transfer mediator that bridges between the anode and the enzyme redox center.
A different approach to assemble biofuel cells is based on the bioelectrocatalyzed oxidation of 1,4-dihydronicotineamide cofactors. Various substrates, for example alcohols, hydroxy acids or sugars undergo biocatalyzed oxidation by enzymes dependent on the NAD(P)+ cofactor (5).
The electrochemical, particularly amperometric biosensors, known in the art are powered by an external power source. This power source is used to apply external voltage to the electrodes and, thus, to polarize the electrodes and to provide electron transfer reactions.
The prior art teaches the use of amperometric biosensing systems as tools to accurately measure biological analytes of interest. However, many problems arise in the application of these biosensors, such as the relative sensitivity, selectivity and stability of the sensing device. In particular, some systems are prone to inaccuracies due to the presence of interfering agents present in the test samples. For example, the biocatalyzed oxidation of glucose is interfered by ascorbic acid or uric acid as contaminants of the analyte.
Thus, there is still a need in the art for biosensors which are highly selective, sensitive, and not prone to interference by other chemicals present in the sample.